Fusion proteins for use as immunogenic enhancers for inducing antigen-specific t cell responses

ABSTRACT

A fusion protein for use as an immunogen enhancer for enhancing antigen-specific T cell responses is disclosed. The fusion protein comprises: (a) an antigen-presenting cell (APC)-binding domain or a CD 91  receptor-binding domain; (b) a protein transduction domain; and (c) an antigen of a pathogen, wherein the APC-binding domain or the CD 91  receptor-binding domain is located at the N-terminus of the fusion protein, and the antigen of the pathogen is located at the C-terminus of the protein transduction domain. The protein transduction domain is selected from the group consisting of: (i) a fusion polypeptide, comprising a T cell sensitizing signal-transducing peptide, a linker, and a translocation peptide; (ii) a T cell-sensitizing signal-transducing peptide; and (iii) a translocation peptide of 34-112 amino acid residues in length.

REFERENCE TO RELATED APPLICATION

This application is a continuation of and claims priority to U.S. Ser.No. 14/095,760, filed Dec. 3, 2013, which status is pending and claimspriority to U.S. Provisional Application Ser. No. 61/733,879, filed Dec.5, 2012, all of which are herein incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The present invention relates generally to fusion proteins, and morespecifically to fusion proteins for enhancing T cell-mediated immuneresponse.

BACKGROUND OF THE INVENTION

Molecular biology has enabled the production of subunit vaccines, inwhich the immunogen is a fragment or subunit of a parent protein orcomplex. The development of a stable vaccine that could elicit T cellsensitizing responses, and be flexible enough to incorporate sequencesfrom many strains of an infectious agent would be desirable

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a fusion protein comprising:

-   -   (a) an antigen-presenting cell (APC)-binding domain or a CD91        receptor-binding domain, located at the N-terminus of the fusion        protein;    -   (b) a protein transduction domain, located at the C-terminus of        the APC-binding domain or the CD91 receptor-binding domain, the        protein transduction domain being selected from the group        consisting of:        -   (i) a fusion polypeptide, comprising a T cell sensitizing            signal-transducing peptide, a linker, and a translocation            peptide, wherein:            -   (1) the T cell sensitizing signal-transducing peptide is                located at the N-terminus of the fusion polypeptide;            -   (2) the linker comprises SEQ ID NO: 15, linking the T                cell sensitizing signal-transducing peptide and the                translocation peptide; and            -   (3) the translocation peptide has 34-112 amino acid                residues in length and comprises the amino acid sequence                that is at least 90% identical to SEQ ID NO: 3, 20 or 4;        -   (ii) a T cell-sensitizing signal-transducing peptide; and        -   (iii) a translocation peptide of 34-112 amino acid residues            in length, comprising the amino acid sequence that is at            least 90% identical to SEQ ID NO: 3, 20 or 4., and    -   (c) an antigen of a pathogen, located at the C-terminus of the        protein transduction domain; wherein:    -   the 1 cell-sensitizing signal-transducing peptide has 28-53        amino acid residues in length and comprises an amino acid        sequence that is at least 90% identical to SEQ ID NO: 31, in        which Xaa⁸ is I or L; Xaa¹⁰ is V, F or A, Xaa¹¹ is M or L, Xaa¹⁷        is L or I; and    -   the APC-binding domain or the CD91 receptor-binding domain is        free of the amino acid sequence of Pseudomonas exotoxin A (PE)        binding domain I if the protein transduction domain is the        translocation peptide (biii).

In one embodiment of the invention, the APC-binding domain or the CD91receptor-binding domain is a polypeptide comprising, an amino acidsequence that is at least 90% identical to the sequence selected fromthe group consisting of SEQ ID NOs: 5, 9, 6, 7, and 8. Alternatively,the APC-binding domain is selected from the group consisting ofreceptor-associated protein-1 (RAP1) domain III, alpha-2-macroglobulinreceptor-associated protein (A2M), HIV-Tat, and heat shock proteins(HSPs), and Pseudomonas exotoxin A (PE) binding domain I.

In another embodiment of the invention, the fusion protein is free ofthe amino acid sequence of Pseudomonas exotoxin A (PE) binding domain I.

In another embodiment of the invention, the fusion protein furthercomprises an endoplasmic reticulum retention sequence located at theC-terminus of the fusion protein.

In another embodiment of the invention, the endoplasmic reticulumretention sequence comprises the amino acid sequence of Lys-Asp-Glu-Leu(SEQ ID NO: 14). The ER retention sequence may comprise a sequenceselected from the group consisting of SEQ ID NOs: 14, 16-19.Alternatively, the ER retention sequence may consist of a sequenceselected from the group consisting of SEQ ID NOs: 16-19.

In another embodiment of the invention, the fusion protein is free of anendoplasmic reticulum retention sequence at C-terminus thereof if theantigen contains 10 or more epitopes.

In another embodiment of the invention, the protein transduction domainis the fusion polypeptide (bi).

In another embodiment of the invention, the protein transduction domainis the T cell-sensitizing signal-transducing peptide (bii).

In another embodiment of the invention, the fusion protein furthercomprises an additional linker between the protein transduction domainand the antigen, the additional linker comprising SEQ ID NO: 15.

In another embodiment of the invention, the protein transduction domainis the translocation peptide (biii).

In another embodiment of the invention, the fusion protein furthercomprises an additional linker between the APC-binding domain or theCD91 receptor-binding domain and the translocation peptide, theadditional linker comprising SEQ ID NO: 15.

In another embodiment of the invention, the protein transduction domaincomprises the sequence of SEQ ID NO: 30.

In another embodiment of the invention, the APC-binding domain comprisesan amino acid sequence that is at least 95% identical to the sequenceselected from the group consisting of SEQ ID NOs: 5, 9, 6, 7, and 8.

In another embodiment of the invention, the APC-binding domain or theCD91 receptor-binding domain is a polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 5, 9, 6, 7,and 8.

In another embodiment of the invention, the T cell sensitizingsignal-transducing peptide comprises an amino acid sequence that is atleast 90% identical to SEQ ID NO: 1 or 2.

In another embodiment of the invention, the T cell sensitizingsignal-transducing peptide comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 1 and 2.

In another embodiment of the invention, the translocation peptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 3, 20, and 4.

In another embodiment of the invention, the translocation peptide has34-61 amino acid residues in length.

In another embodiment of the invention, the protein transduction domainof the fusion protein as aformentioned possesses the following features:(i) the T cell-sensitizing signal-transducing peptide comprises theamino acid sequence of SEQ. ID NO: 1 or 2; and (ii) the translocationpeptide comprises the amino acid sequence that is at least 95% identicalto SEQ ID NO: 3.

The T cell sensitizing signal-transducing peptide exhibits acharacteristic of eliciting an antibody that recognizes and binds to theamino acid sequence of K¹(X)²E³(X)⁴(X)⁵Y⁶P⁷P⁸P⁹Y¹⁰ (SEQ ID NO: 32) ofCD28 receptor on T cells, wherein (X)² is I or L; (X)⁴ is V, F or A, and(X)⁵ is M or L.

In another aspect, the invention relates to a fusion protein consistingof:

-   -   (a) an antigen-presenting cell (APC)-binding domain or a CD91        receptor-binding domain, located at the N-terminus of the fusion        protein;    -   (b) a protein transduction domain, located at the C-terminus of        the APC-binding domain or the CD91 receptor-binding domain, the        protein transduction domain being selected from the group        consisting of:        -   (i) a fusion polypeptide, comprising a T cell sensitizing            signal-transducing peptide, a linker, and a translocation            peptide, wherein:            -   (1) the T cell sensitizing signal-transducing peptide is                located at the N-terminus of the fusion polypeptide;            -   (2) the linker comprises SEQ ID NO: 15, linking the T                cell sensitizing signal-transducing peptide and the                translocation peptide; and            -   (3) the translocation peptide has 34-112 amino acid                residues in length and comprises the amino acid sequence                that is at least 90% identical to SEQ ID NO: 3, 20 or 4;        -   (ii) a T cell-sensitizing signal-transducing peptide; and        -   (iii) a translocation peptide of 34-112 amino acid residues            in length, comprising the amino acid sequence that is at            least 90% identical to SEQ ID NO: 3, 20 or 4; and    -   (c) an antigen of a pathogen, located at the C-terminus of the        protein transduction domain; wherein:    -   the T cell-sensitizing signal-transducing peptide has 28-53        amino acid residues in length and comprises an amino acid        sequence that is at least 90% identical to SEQ II) NO: 31, in        which Xaa⁸is I or L.; Xaa¹⁰ is V, F or A, Xaa¹¹ is M or L, Xaa¹⁷        is L or I; and    -   the APC-binding domain or the CD91 receptor-binding domain is        free of the amino acid sequence of Pseudomonas exotoxin A (PE)        binding domain I if the protein transduction domain is the        translocation peptide (biii).

The antigen-presenting cell (APC) may be selected from the groupconsisting of dendritic cells, macrophages, B-cells and monocytes.

In one embodiment of the invention, the cell membrane of the APCcomprises a CD91 receptor.

In another aspect, the invention relates to a vaccine compositioncomprising: (a) a therapeutically effective amount of a fusion proteinas aforementioned; and (b) an adjuvant.

The adjuvant is either an antigen delivery agent or an immunepotentiator. In one embodiment of the invention, the vaccine compositioncomprises an antigen delivery agent and is free of an immunepotentiator.

Further in another aspect, the invention relates to a method forinducing enhanced pathogen antigen-specific T cell responses,comprising: administering a vaccine composition comprising atherapeutically effective amount of a fusion protein as aforementionedto a subject in need thereof, and thereby inducing enhanced pathogenantigen-specific T cell responses.

Further in another aspect, the invention relates to a method for killinga disease cell that presents an antigen via class I MHC molecules on thecell membrane of the disease cell, comprising: administering a vaccinecomposition comprising a therapeutically effective amount of a fusionprotein as aforementioned to a subject in need thereof, and therebykilling the disease cell that that presents the antigen via class I MHCmolecules on the cell membrane of the disease cell.

In one embodiment of the invention, the disease cell is a cancer cell.

Yet in another aspect, the invention relates to a method for preventing,treating infection caused by a pathogen, and/or minimizing symptomscaused by the infection, comprising: administering a vaccine compositioncomprising a therapeutically effective amount of the fusion protein ofclaim 1 to a subject in need thereof, and thereby preventing, treatinginfection caused by the pathogen, and/or minimizing symptoms caused bythe infection.

The pathogen may be at least one selected from the group consisting ofHuman Papillomavirus (HPV), Porcine Reproductive and RespiratorySyndrome Virus (PRRSV), Human Immuno-deficient Virus (HIV-1), flu virus,Dangue virus, Hepatitis C virus (HCV). Hepatitis B virus (HBV) andPorcine Circovirus 2 (PCV2).

In one embodiment of the invention, the fusion protein as aforementionedis for use in enhancing an antigen-specific cytotoxic T cell response ina subject in need thereof. The fusion protein may also be for use inenhancing an antigen-specific CD4+ T cell response, or for use as animmunogenic enhancer for inducing an enhanced antigen-specific antibodytiter response, in a subject in need thereof.

These and other aspects will become apparent from the followingdescription of the preferred embodiment taken in conjunction with thefollowing drawings, although variations and modifications therein may beaffected without departing from the spirit and scope of the novelconcepts of the disclosure.

The accompanying drawings illustrate one or more embodiments of theinvention and, together with the written description, serve to explainthe principles of the invention. Wherever possible, the same referencenumbers are used throughout the drawings to refer to the same or likeelements of an embodiment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a vector map.

FIG. 2 is a photograph showing the result of SDS-PAGE analyses of fusionproteins.

FIG. 3 is a vector map.

FIG. 4 is a schematic drawing illustrating one embodiment of theinvention.

FIG. 5A shows immunization schedules.

FIG. 5B is a photograph showing the result of SDS-PAGE analyses offusion proteins.

FIGS. 5C-D are graphs showing tumor size curves and percentage oftumor-free mice in the animal groups vaccinated with various fusionproteins or placebo, respectively.

FIG. 6A shows a flow chart for preparation of fusion proteins.

FIG. 6B is a photograph showing the result of SDS-PAGE analyses offusion proteins.

FIG. 7 is a schematic drawing illustrating the mechanisms of actions ofT cell-sensitizing fusion proteins.

FIG. 8 shows sequence alignments of CD28 from various species.

FIG. 9A shows immunization schedules.

FIGS. 9B-C show tumor size curves and survival rate in the animal groupsvaccinated with various fusion proteins or placebo, respectively.

FIG. 10 is a schematic drawing showing a RAP1-containing vector used forgenerating a plasmid containing a DNA insert from a pathogen.

FIG. 11 are schematic drawings illustrating constructions of fusionproteins containing antigens of various pathogens.

FIGS. 12A-F are photographs showing the results of SDS-PAGE analyses ofvarious fusion proteins.

FIGS. 13A-B show animal groups, vaccines and dosage used for immunizingthe animals, and immunization schedules.

FIGS. 14A-D are tables showing the results of ex vivo antigen-specificimmune response analyses of CO3+/CD4+ splenocytes and CD3+/CD8+splenocytes from the animal groups of FIG. 13A vaccinated with placeboor a fusion protein containing E7₁₆, E7₁₈. HCVcore, or HBx antigen.

FIGS. 15A-J show IFNγ+ cell counts in ex vivo antigen-specific immuneresponse analyses of CD3+/CD8+ splenocytes and CD3+/CD4+ splenocytesfrom the animal groups of FIG. 13A vaccinated with placebo or a fusionprotein containing a PCV2 (15A-B) or PRRSV antigen (15C-J).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is more particularly described in the followingexamples that are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art. Various embodiments of the invention are now described indetail. Referring to the drawings, like numbers indicate like componentsthroughout the views. As use din the description herein and throughoutthe claims that follow, the meaning of “a”, “an”, and “the” includesplural reference unless the context clearly dictates otherwise. Also, asused in the description herein and throughout the claims that follow,the meaning of “in” includes “in” and “on” unless the context clearlydictates otherwise. Moreover, titles or subtitles may be used in thespecification for the convenience of a reader, which shall have ininfluence on the scope of the present invention. Additionally, someterms used in this specification are more specifically defined below.

Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used. Certain terms that are used todescribe the invention are discussed below, or elsewhere in thespecification, to provide additional guidance to the practitionerregarding the description of the invention. For convenience, certainterms may be highlighted, for example using italics and/or quotationmarks. The use of highlighting has no influence on the scope and meaningof a term; the scope and meaning of a term is the same, in the samecontext, whether or not it is highlighted. It will be appreciated thatsame thing can be said in more than one way. Consequently, alternativelanguage and synonyms may be used for any one or more of the termsdiscussed herein, nor is any special significance to be placed uponwhether or not a term is elaborated or discussed herein. Synonyms forcertain terms are provided. A recital of one or more synonyms does notexclude the use of other synonyms. The use of examples anywhere in thisspecification including examples of any terms discussed herein isillustrative only, and in no way limits the scope and meaning of theinvention or of any exemplified term. Likewise, the invention is notlimited to various embodiments given in this specification.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In the case of conflict, thepresent document, including definitions, will control.

As used herein, “around”, “about” or “approximately” shall generallymean within 20 percent, preferably within 10 percent, and morepreferably within 5 percent of a given value or range. Numericalquantities given herein are approximate, meaning that the term “around”,“about” or “approximately” can be inferred if not expressly stated.

The term “an antigen-presenting cell (APC) or accessory cell” refers toa cell that displays foreign antigens complexed with majorhistocompatiblity complexes (MHC's) on their surfaces. T-cells mayrecognize these complexes using their T-cell receptors (TCRs). Thesecells process antigens and present them to T-cells. Main types ofprofessional antigen-presenting cell: dendritic cells (DCs),macrophages, monocytes, and certain B-cells.

The term “an antigen-presenting cell (APC)-binding domain” refers to adomain that can bind to an antigen-presenting cell (APC). TheAPC-binding domain may be a polypeptide comprising an amino acidsequence that is at least 90% identical to the sequence selected fromthe group consisting of SEQ ID NOs: 5, 6, 7, 8, and 9. An APC-bindingdomain is a ligand that recognizes and binds to a receptor on APC.

Cluster of differentiation 91 (CD91) is a protein that forms a receptorin the membrane of cells and is involved in receptor-mediatedendocytosis.

The term “a protein transduction domain” refers to a polypeptide or afusion polypeptide having a function to sensitize T-cells and thusenhance antigen-specific T cell responses, and/or to guide or direct anantigen toward (i.e., to target to) class I major histocompatibilitycomplex (MHC-I) pathway (i.e., a cytotoxic T cell pathway) of antigenpresentation.

The term “to sensitize T cells” generally means that CD8+ and CD4+ Tcells are sensitized and as a result, CD8+ (CTL) and CD4+ T cellresponses to an antigen challenge are enhanced. An antigen-specific cellmediated immune response is measured by quantifying the production ofantigen-specific induced γ-interferon in response to an antigen. Forexample, without a sensitization signal (i.e., without the proteintransduction domain), an antigen alone may induce weak or no cellmediated immune response at all, i.e., weak or no production ofantigen-specific γ-interferon from CD8+ and CD4+ T cells, while in thepresence of a sensitization signal (the protein transduction domain),the antigen may induce an enhanced cell mediated immune response. Thus,the function of a sensitization signal (the protein transduction domain)is to sensitize CD4+ and CD8+ T cells in a host so that when the host islater challenged by an antigen, the antigen can induce an enhancedantigen-specific cell mediated immune response due to prior CD4+ andCD8+ T cell sensitization.

A protein transduction domain may be a peptide and/or polypeptideselected from the group consisting of:

-   -   (i) a fusion polypeptide, comprising a T cell sensitizing        signal-transducing peptide, a linker, and a translocation        peptide, wherein:        -   (1) the T cell sensitizing signal-transducing peptide is            located at the N-terminus of the fusion polypeptide;        -   (2) the linker comprises SEQ ID NO: 15, linking the T cell            sensitizing signal-transducing peptide and the translocation            peptide; and        -   (3) the translocation peptide has 34-112 amino acid residues            in length and comprises the amino acid sequence that is at            least 90% identical to SEQ ID NO: 3, 20 or 4;    -   (ii) a T cell-sensitizing signal-transducing peptide; and    -   (iii) a translocation peptide of 34-112 amino acid residues in        length, comprising the amino acid sequence that is at least 90%        identical to SEQ ID NO: 3, 20 or 4.

A protein transduction domain may be “a fusion polypeptide”, in whichthe fusion polypeptide comprises a T cell sensitizing signal-transducingpeptide, a linker, and a translocation peptide. For example, the fusionpolypeptide may be the polypeptide “CD28convPE_(t)”.

The term “CD28conv” refers to a CD28 conserved region, which is a “Tcell sensitizing signal-transducing peptide”. It's an epitope forinducing CD28 agonist antibody.

The term “PE_(t)” refers to a translocation peptide with 34-112 aminoacid residues in length.

A linker is present between the “CD28conv” and the “PE_(t)”. Theorientation or arrangement of the fusion polypeptide “CD28convPE_(t)” isimportant in that “CD28conv” (or the T cell sensitizingsignal-transducing peptide) must be at the upstream to the PE_(t) (orthe translocation peptide), i.e., PE_(t) must be at the C-terminus ofthe “CD28conv” to obtain enhanced T-cell responses. The “CD28convPE_(t)”can raise much higher IgG titer (called CD28-specific agonist antibody)specific to CD28conv than the reversed orientation fusion peptidePE_(t)CD28conv. The CD28-specific agonist antibody can sensitize bothCD4+ and CD8+ T cells. The correct orientation fusion polypeptideCD28convPE_(t) contains a linker (RXRXKR) between CD28conv and PE_(t)domains. The linker contains an antigen presenting cell (APC)-specificprotease (cathepsin L) cutting site Lys-Arg (KR). Therefore, the fusionprotein RAP1-CD28convPE_(t)-Antigen-K3 can be digested into the twofragments: RAP1-CD28conv and PE_(t)-Antigen-K3. The RAP1-CD28convfragment can be further digested in the lysosome and the epitope ofCD28conv is then presented to the APC cell surface via MHC II pathway,which in turn elicits a humoral immune response producing CD28 agonistantibody. Thus, CD28 agonist antibody is produced by B cells. This CD28agonist antibody can bind to CD28 on the T cell surface and pre-activatethe T cells (CD4+ and CD8+ T cells).

A “T cell-sensitizing signal-transducing peptide” has 28-53 amino acidresidues in length and comprises an amino acid sequence that is at least90% identicial to SEQ ID NO: 31, in which Xaa⁸ is I or L; Xaa¹⁰ is V, For A, Xaa¹¹ is M or L, Xaa¹⁷ is L or I.

The T cell-sensitizing signal-transducing peptide comprises the criticalregion K¹(I/L)²E³(V/F/A)⁴(M/L)⁵Y⁶P⁷P⁸P⁹Y¹⁰ (SEQ ID NO: 32), wherein (X)²is I or L; (X)⁴ is V, F or A, (X)⁵ is M or L.

A T cell sensitizing signal-transducing peptide(TDIYFCKIEFMYPPPYLDNEKSNGTIIH; SEQ ID NO: 31, wherein X⁸ is I, X¹⁰ is F,X¹¹ is M) specific for mice was used in the following examples.

FIG. 7 is a schematic drawing illustrating the mechanisms of actions ofT cell-sensitizing fusion proteins using the fusion proteinRAP1-CD28convPE_(t)-E7-K3 as an example. RAP1-CD28convPE_(t)-E7-K3comprises from N-terminus to the C-terminus: (1) the domain III of thefull length RAP1 at the N-terminus, (2) CD28conv, (3) a linker, (4) amodified translocation peptide from Pseudomonas Exotoxin A, (5) a fulllength HPV type 16 E7 protein, and (6) the triple KDEL as an ERretention signal at the C-terminus. The HPV16 E7 protein is processedinto epitopes within the cells of a subject immunized with the fusionprotein. The RAP1-CD28convPE_(t)-E7-K3 can elicit a better cytotoxicT-cell (CTL) response than the traditional vaccines that contain antigenonly. RAP1-CD28convPE_(t)-E7K3 protein was designed to improve APC (suchas dendritic cell) uptake efficiency and enhance HPV16 E7 antigenprocessing toward a proteasome pathway, then presented via MHC Icomplexes. The mechanism of action of HPV16 E7 protein-specific CTLimmune response elicited by the vaccine RAP1-CD28convPE_(t)-E7-K3 isillustrated in FIG. 7: (a) the vaccine binds to APC (such as dendriticcell) surface receptor (CD91) and internalized via endocytosis; (b1)RAP1-CD28convPE_(t)-E7-K3 undergoes proteolytic hydrolysis by cathepsinL protease digestion at the site before the the translocation peptidePE_(t); (b2) or recycles to the E.R. and undergoes proteolytichydrolysis by furin protease at the site before the translocationpeptide PE_(t); (b3) At the meantime, RAP1-CD28conv was digested bylysosomal protease and the epitopes of CD28conv are then presented tothe cell surface via MHC II and elicits CD28 agonist antibodyproduction, which can pre-active T cells; (c) The most important step istramsmembrane translocation of the PE_(t)-E7-K3 into the cytoplasmiccompartment from the lysosome by translocation peptide (PE_(t)); (d) thePE_(t)-E7-K3 undergoes digestion via proteasome pathway, then theepitopes of E7 are presented by MHC I complex and elicit E7-specificcell mediated immune response.

FIG. 8 shows a sequence alignment of CD28 conserved regions from variousspecies and the consensus sequence. The underlined sequence (KIEVMYPPY;SEQ ID NO: 32, where X² is I, X⁴ is V, X⁵ is M) within the consensussequence is a critical region for CD28 agonist antibody recognition andbinding. This critical region sequence can be represented byK¹(I/L)²E³(V/F/A)⁴(M/L)⁵Y⁶P⁷P⁸P⁹Y¹⁰, in which only the fourth amino acidresidue therein is species-specific and should be V in human, rat,porcine, bovine, sheep, dog and horse; F in mouse, and V in turkey. Thecritical region sequence may be represented asK¹(X)²E³(X)⁴(X)⁵Y⁶P⁷P⁸P⁹Y¹⁰ (SEQ ID NO: 32), wherein (X)² is I or L;(X)⁴ is V, F or A, (X)⁵ is M or L.

PE_(t) may comprise the amino acid sequence that is at least 90%identical to SEQ ID NO: 3 or 20. For example, the amino acid sequence ofPE_(t) may be a.a. 280-a.a. 313 (SEQ ID NO: 3), a.a. 268-a.a. 313 (SEQID NO: 20), a.a. 253-a.a. 313, or a.a. 253-a.a. 364 (SEQ. ID NO: 4) ofPE. That is, the amino acid sequence of PE_(t) may contain any region ofthe PE domain II (a.a. 253 to a.a. 364; SEQ ID NO: 4) as long as itcomprises a.a. 280-a.a. 313 (SEQ ID NO: 3) essential fragment.

An antigen may be a pathogenic protein, polypeptide or peptide that isresponsible for a disease caused by the pathogen, or is capable ofinducing an immunological response in a host infected by the pathogen,or tumor-associated antigen (TAA) which is a polypeptide specificallyexpressed in tumor cells. The antigen may be selected from a pathogen orcancer cells including, but not limited to, Human Papillomavirus (HPV),PRRSV, HIV-1, flu virus. Dangue virus, Hepatitis C virus (HCV).Hepatitis B virus (HBV), Porcine Circovirus 2 (PCV2), non-small celllung cancer, breast carcinoma, melanoma, lymphomas, colon carcinoma,hepatocellular carcinoma and any combination thereof. For example, HPVE7 protein (E7), HCV core protein (HCV core), HBV X protein (HBx) wereselected as antigens for vaccine development. The antigen may be afusion antigen from a fusion of two or more antigens selected from oneor more pathogenic proteins. For example, a fusion antigen of PRRSV ORF6and ORF5 fragments, or a fusion of antigenic proteins from PRRSV andPCV2 pathogens.

The function of an endoplasmic reticulum retention sequence is to assisttranslocation of an antigen from an endocytotic compartment into ER andretains it in the lumen. It comprises the sequence Lys Asp Glu Leu(KDEL) or RDEL. An ER sequence may comprise, or consists essentially of,or consist of, the sequence of KKDLRDELKDEL (SEQ ID NO: 16),KKDELRDELKDEL (SEQ ID NO: 17), KKDELRVELKDEL (SEQ ID NO: 18).

Receptor-associated protein (RAP1) with a molecular weight of 39 kDa isan ER resident protein and molecular chaperone for LDL receptor-relatedprotein. It has a high binding affinity to CD91 (Kd˜ 3 nM) and iscomposed by three functional-similar domains.

The invention relates to the discovery of induction and enhancement of Tcell mediated immune responses by fusion proteins according to theinvention. Using RAP1-CD28convPE_(t)-E7-K3 an example, the strategy ofRAP1-CD28convPE_(t)E7-K3 vaccine is focused primarily on stimulating theproduction and activation of T cells that can recognize HPV16 infectedcells expressing the target antigen E7. By delivering antigens todendritic cells, it can generate antigen-specific CD8+T cells and CD4+ Tcells. Type 1-helper CD4+ cells particularly are able to efficientlystimulate and augment the immune response of cytotoxic CD8+ T cells.Together, these two arms of the adaptive immune system have thespecificity and potency to kill HPV16-infected cells or HPV16-associatedtumor cells at multiple sites in the body without inflicting significantdamage on normal tissues.

The term “subject” refers to a human or a non-human animal.

The term “treating” or “treatment” refers to administration of aneffective amount of the fusion protein to a subject in need thereof, whohas cancer or infection, or a symptom or predisposition toward such adisease, with the purpose of cure, alleviate, relieve, remedy,ameliorate, or prevent the disease, the symptoms of it, or thepredisposition towards it. Such a subject can be identified by a healthcare professional based on results from any suitable diagnostic method.

The term “an effective amount” refers to the amount of an activecompound that is required to confer a therapeutic effect on the treatedsubject. Effective doses will vary, as recognized by those skilled inthe art, depending on rout of administration, excipient usage, and thepossibility of co-usage with other therapeutic treatment.

Abbreviations: CD 28, Cluster of Differentiation 28.

EXAMPLES

Without intent to limit the scope of the invention, exemplaryinstruments, apparatus, methods and their related results according tothe embodiments of the present invention are given below. Note thattitles or subtitles may be used in the examples for convenience of areader, which in no way should limit the scope of the invention.Moreover, certain theories are proposed and disclosed herein; however,in no way they, whether they are right or wrong, should limit the scopeof the invention so long as the invention is practiced according to theinvention without regard for any particular theory or scheme of action.

Example 1 Constructions of Expression Vectors

FIG. 1 shows the expression vector for the fusion proteinRAP1-PE₂₆₈₋₃₁₃-E7-K3, which comprises a RAP1 domain 3 (SEQ ID NO: 5), atranslocation minimum essential peptide (PE₂₆₈₋₃₁₃; SEQ ID NO: 20), theantigen E7, and an endoplasmic reticulum retention sequence (K3, or aKDEL signal; SEQ ID NO: 16, 17, 18, or 19). The translocation minimumessential peptide (PE₂₆₈₋₃₁₃; SEQ ID NO: 20) was obtained from PE (SEQID NO: 10) polypeptide sequence region from a.a. 268 to a.a. 313.

This expression vector was constructed by using the plasmid RAP1-K3shown in FIG. 10. The plasmid RAP1K-3 (FIG. 10), which contains a fusiongene of RAP-1 domain 3 and K3, was generated as follows: A DNA fragmentencoding ^(Ndef)RAP1-^((EcoRI, Xhol))-K3^(Xhol) was synthesized by a PCRmethod and ligated into the plasmid pUC18 backbone with kanamycinresistance gene to obtain the plasmid RAP1-K3 (FIG. 10). To generate theexpression vector RAP1-PE₂₆₈₋₃₁₃-E7-K3 (FIG. 1), a DNA fragment encodingPE₂₆₈₋₃₁₃-E 7 was inserted into the plasmid RAP1-K3 (FIG. 10). Usingsimilar methods, DNA fragments encoding various fusion peptides weregenerated by PCR (FIG. 11) and inserted, respectively, into the plasmidRAP1-K3 (FIG. 10) to generate expression vectors for various fusionproteins (FIGS. 12A-F). FIG. 3 illustrates the expression vector for thefusion protein RAP1-CD28convPE_(t)-E7-K3 (FIG. 3).

The fragment CD28convPE_(t) contains cathepsin L and furin proteasecutting sites. FIG. 4 shows an amino acid sequence map of the fusionprotein RAP1-CD28convPE_(t)-E7-K3 to illustrate the importance of thefragment CD28convPE_(t). The two arrows indicate cathepsin L and furinprotease cutting sites, respectively. These two cutting sites not onlyserve as linkers to ligate CD28conv and PE_(t)-E7 but also allow cuttingof the fusion protein to release the fragment PE_(t)-E7-K3 to thecytoplasm from the lysosome. Any other antigens of interest from variouspathogens may replace E7 to fuse with CD28convPE_(t) and the fusionproduct can then be inserted into the plasmid of FIG. 10 to make avariety of expression vectors for fusion proteins illustrated in FIG.11.

The sequence of PE₂₆₈₋₃₁₃ is:pletftrhrqprgweqleqcgypvqrlvalylaarswnqvdqvir (SEQ ID NO: 20). The wholesequence of CD28convPE_(t) is as follows:tdiyfckiefmypppyldneksngtiihrarykrgweqleqcgypvqrlvalylaarlswnqvdqvirgs(SEQ ID NO: 30), the sequence underlined represents a linker sequencecontaining Cathepsin L and furin protease cutting sites.

Example 2 Protein Expression

E. coli BL21 cells harboring a protein expression vector were culturedin Luria Bertani broth containing 25 μg/ml kanamycin at 37° C. When theculture reached an early log phase, (A600=0.1 to 0.4),isopropyl-1-thio-β-D-galactopyranoside (IPTG) was added at a finalconcentration of 0.5 to 2 mM for induction. Cells were harvested after 4hours IPTG induction and disrupted by sonication. The overexpressedprotein-containing inclusion bodies were isolated and solubilized in 8Murea/TN buffer (8M urea, 50 mM Tris, 50 mM NaCl, pH 8.0).

The refolding of the fusion protein RAP1-PE₂₆₈₋₃₁₃-E7-K3 was per formedby dialysis against 50X volume of TNZ buffer (50 mM Tris, 50 mM NaCl and0.01 mM ZnCl₂, pH 8.0) at 4° C. overnight. The refolded proteins weresubject to SDS-PAGE analyses under reduced (with dithiothreitol; +DTT)and non-reduced (without dithiothreitol; −DTT) conditions (FIG. 2). Theresults indicated that most of the refolded proteins were monomers undera non-reduced condition, indicating that the RAP1 fusion proteinrefolded easily and were not aggregated (FIG. 2).

FIG. 6A is a flow chart illustrating that the fusion proteinsRAP1-CD28PE_(t)-E7-K3 (with mouse CD28conv or human (CD28conv) wereexpressed and extracted from the inclusion bodies of E. coli cells).SDS-PAGE analyses indicated that the fusion proteins refolded well (FIG.6B).

FIG. 11 illustrates a list of fusion proteins that were expressed usingsimilar method described above: (1) RAP1-PE₂₆₈₋₃₁₃-E7-K3; (2)RAP1-CD28convPE_(t)-E7-K3; (3) RAP1-CD28PE_(t)-E7₁₈-K3; (4)RAP1-HCVcore-K3; (5) RAP1-CD28conv-HCVcore-K3;(6)RAP1-CD28convPE_(t)-HCVcore-K3; (7) RAP1-HBX; (8) RAP1-HBx-K3; (9)RAP1-CD28conv-HBx; (10) RAP1-CD28conv-HBx-K3; (11)RAP1-CD28convPE_(t)-HBx; (12) RAP1-CD28convPE_(t)-HBx-K3; (13)RAP1-PCV2_(ORF2)-K3; (14) RAP1-PE₂₆₈₋₃₁₃₋PCV2_(ORF2)-K3; (15)RAP1-CD28convPE_(t)-PCV2_(ORF2)-K3: (16) RAP1-PE₂₆₈₋₃₁₃-DGD-K3; (17)RAP1-PE₂₆₈₋₃₁₃-M12-K3; (18) RAP1-PE₂₆₈₋₃₁₃-PQAB-K3; (19)RAP1-PE₂₆₈₋₃₁₃-RSAB-K3; (20) RAP1-CD28convPE_(t)-DGD-K3; (21)RAP1-CD28convPE_(t)-M12-K3; (22) RAP1-CD28convPE_(t)-PQAB-K3; (23)RAP1-CD28convPE_(t)-RSAB-K3. These fusion proteins were refolded usingthe same method described above. The results of SDS-PAGE analysesindicated these fusion proteins al refolded well and were thus used forpreparing vaccines (FIGS. 12A-F).

Example 3 RAP1-PE₂₆₈₋₃₁₃-E7-K3 Inhibits Growth of Tumors Induced byHuman Papilloma Virus (HPV) Type 16 E7 Protein

The fusion proteins PE₄₀₇-E7-K3 and RAP1-PE₂₆₈₋₃₁₃-E7-K3 were expressedas described above and protein refolding examined by SDS-PAGE (FIG. 5B).Mice were challenged with 2×10³ TC-01 cells (a mouse lung epithelia cellline harboring HPV type 16 E7 gene) via s.c. injection to induce HPV-16type carcinoma. Twelve days after the TC-01 cell challenge, mice werevaccinated via s.c. with placebo (PBS+aluminum phosphate), PE407-E7-K3(200 μg/dose) or RAP1-PE₂₆₈₋₃₁₃-E7-K3 (200 μg/dose) with AS04C(GlaxoSmithKline) as an adjuvant once per week for 3 weeks (FIG. 5A).AS04C, which is a cytotoxic T lymphocyte-enhacing adjuvant, comprisesMPL (monophosphoryl lipid A, an immune potentiator) and aluminumphosphate (a protein absorbent for antigen delivery). The term “K3”stands for the amino acid sequence KDELKDELKDEL (SEQ. ID NO: 19). Thesize of tumors and the number of tumor-free animals in each group wererecorded (FIGS. 5C-D). The tumor growth was significantly suppressed byboth vaccines PE₄₀₇-E7-K3 and RAP1-PE₂₆₈₋₃₁₃-E7-K3 with AS04C as anadjuvant. However, the mouse group vaccinated with RAP1-PE₂₆₈₋₃₁₃-E7-K3had a higher rate of tumor-free mice. This indicated that the vaccineRAP1-PE₂₆₈₋₃₁₃-E7-K3 was as effective as or better than PE₄₀₇-E7-K3 insuppressing tumor growth, however was better in increasing thepercentage of tumor-free animals.

Example 4 RAP1-CD28convPE_(t)-E7-K3 Inhibits Growth of Tumors Induced byHuman Papilloma Virus (HPV) Type 16 E7 Protein and Increases SurvivalRate

The effects of the fusion proteins PE₄₀₇-E7-K3 andRAP1-CD28convPE_(t)-E7-K3 with or without an immune potentiator on tumorsize and survival rate were examined. Mice were challenged with a higherdose of TC-01 cells (3×10⁴) via s.c. injection. Seven days after thechallenge, mice were vaccinated via s.c. with placebo, PE₄₀₇-E7-K3 (100μg/dose) or RAP1-CD28convPE_(t)-E7-K3 (100 μg/dose) with the immunepotentiator GPI-0100 or the protein absorbent aluminum phosphate onceper week for 3 weeks (FIG. 9A). GPI-0100 is a Th1/CTL stimulatingadjuvant (immune potentiator). The size of the tumors and the survivalrate in each group were recorded (FIGS. 9B-C). When combined with theadjuvant GPI-0100, both PE₄₀₇-E7-K3 and RAP1-CD28convPE_(t)-E7-K3suppressed the tumor growth. Unexpectedly, it was discovered that theeffect of RAP1-CD28convPE_(t)-E7-K3 in inhibiting tumor growth was notdependent on the adjuvant. When combined with the absorbent aluminumphosphate rather than with the adjuvant GPI-0100,RAP1-CD28convPE_(t)-E7-K3 could still significantly suppress the tumorgrowth with the same potency as that when combined with the immunepotentiator GPI-0100 (FIG. 9B, unfilled triangle v. filed reversetriangle).

In contrast, the potency of PE₄₀₇-E7-K3 in suppressing the tumor growthdepended on the adjuvant. When combined with the absorbent aluminumphosphate, PE₄₀₇-E7-K3 became less potent than that when combined withthe immune potentiator GPI-0100 (FIG. 9B solid square v. unfilledcircle).

On the other hand, mice administrated with RAP1-CD28convPE_(t)-E7-K3 incombination with the immune potentiator GPI-0100 or the absorbentaluminum phosphate had a better survival rate than the groups vaccinatedwith PE₄₀₇-E7-K3 in combination with GPI-0100 or aluminum phosphate(FIG. 9C). This indicated that RAP1-CD28convPE_(t)-E7-K3 could elicitTh1/CTL immune responses even without the immune potentiator GPI-0100.The results also indicated that the fusion proteinRAP1-CD28convPE_(t)-E7-K3 was superior to PE₄₀₇-E7-K3 as a vaccine forincreasing the survival rate of the animals.

Example 5 Immunogenicity Assays

The immunogenicities of various vaccines were tested. Briefly, mice weredivided into the following groups: HPV16 E7, HPV 18 E7, HCV core, HBVHBx, PCV2ORF2 and PRRSV (FIG. 13A). Each group was further divided intosubgroups, and each subgroup was injected with either a placebo or avaccine designed to target toward a certain antigen, or certainantigens, of a pathogen via s.c. once per week for 3 weeks (FIG. 13B).Except the vaccines targeted to PRRSV, each vaccine was composed of asingle fusion protein and the absorbent aluminum phosphate, in which thesingle fusion protein contained at least an antigen of a pathogen. Theantigen was either a full-length protein from a pathogen, or anon-full-length protein that contained at least one epitope of anantigen of a pathogen, or was a fusion peptide of two or more antigens,in which each of the antigens was selected from different proteins of apathogen.

The immunization schedule, vaccines and does are illustrated in FIGS.13A-B. Briefly, mice were vaccinated once per week for 3 weeks withvaccines listed in FIG. 13A. All mice were sacrificed 7 days after thelast immunization, and the spleens were harvested. Splenocytes wereisolated and cultured in 6-well plate (2×10⁷ cells/2 ml/well) with 10μg/ml of respective recombinant antigens of pathogens to stimulate thesplenocytes in the presence of 1 μg/ml GolgiPlug (BD Pharmingen, SanDiego, Calif.) at 37° C. for 16 hr.

The stimulated splenocytes were washed with FACScan buffer and the cellsurface markers CD8a, CD4, and CD3 were stained withphycoerythrin-conjugated monoclonal rat anti-mouse CD8a,AF700-conjugated monoclonal rat anti-mouse CD4 and AF647-conjugatedmonoclonal rat anti-mouse CD3 antibodies. The cells were thenpermeabilized and fixed by Cytofix/Cytoperm kit according to themanufacturer's instructions (BD Pharmingen). Intracellular IFN-γ wasstained with AF488-conjugated rat anti-mouse IFN-65 to measure theimmune response and cytokine levels. Flow cytometry analyses wereperformed using Gallios flow cytometry with Kaluza analysis software(Beckman Coulter).

The following PRRSV vaccines were tested for immunogenicities:PE₄₀₇-PRRSV-K3, RAP1-PE₂₆₈₋₃₁₃PRRSV-K3 or RAP1-CD28convPE_(t)-PRRSV-K3vaccine. Each vaccine contained a mixture of four different fusionproteins, and each fusion protein contained a different antigen that isselected from the group consisting of DGD, M12, PQAB and RSAB (FIG.13A). Vaccination of mice and stimulations of splenocytes were performedusing similar method as described above. Briefly, all mice weresacrificed 7 days after the last immunization, and spleens wereharvested. The splenocytes were isolated and cultured in 6-well plate(2×10⁷ cells/2 ml/well) with 10 μg/ml of the recombinant DGD, M12, PQABor RSAB antigens, separately, to stimulate the splenocytes in thepresence of 1 μg/ml GolgiPlug (BD Pharmingen, San Diego, Calif.) at 37°C. for 16 hr.

The amino acid sequence of “DGD” (SEQ ID NO: 26) is as follows:

RHHFTPSERQLCLSSIQTAFNQGAGTCILSDSGRISYTVEFSLPTHHTVR LIRVTAPPSALDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANADVVSLTCPVAAGECAGPADSGDALLERNYPTG AEFLGDGGDVRHHFTPSERQLCLSSIQTAFNQGAGTCILSDSGRISYTVE FSLPTHHTVRLIRVTAPPSA,DGD represents a fusion antigen of PRRSV ORF7 a.a. 64-a.a. 123(boldface), linker (underlined) and ORF7 a. a. 64-a.a. 123 (boldface).

The term “M12” represents a antigen of PRRSV ORF1b a.a. 1046-a.a. 1210.Its amino acid sequence (SEQ ID NO: 27) is as follows:

NNKECTVAQALGNGDKFRATDKRVVDSLRAICADLEGSSSPLPKVAHNLGFYFSPDLTQFAKLPIELAPHWPVVSTQNNEKWPDRLVASLRPLDKNSRACIGAGYMVGPSVFLGTPGVVSYYLTKFVKGEAQVLPETVFSTGRIEVDCRE YLDDREREVAASLPH,

The amino acid sequence of “PQAB” (SEQ ID NO: 28) is as follows:

GSSLDDFCYDSTAPQKVLLAFSITYASNDSSSHLQLIYNLTLCELNGTDW LANKFDWA,PQAB represents a fusion antigen of PRRSV American strain ORF6 a.a.2-a.a. 26 and ORF5 a.a. 31-a.a. 63 (underlined).

The amino acid sequence of RSAB is

(SEQ ID NO: 29) MGSLDDFCNDSTAAQKLVLAFSITYTPIFVAGGSSSTYQYIYNLTICELNGTDWLSNHFDWA,The term “RSAB” represents a fusion antigen of PRRSV European strainORF6 a.a. 2-28 and ORF5 a.a. 31-64 (underlined).

Example 6

The fragment of RAP1 domain 3 of the fusion proteinRAP1-CD28convPE_(t)-E7-K3 is replaced by A2M minimum (SEQ ID NO: 6),HIV-Tat minimum (SEQ ID NO: 7) or HSPs minimum (SEQ ID NO: 8) togenerate the fusion proteins A2M-CD28convPE_(t)-E7-K3,Tat-CD28convPE_(t)-E7-K3 and HSP-CD28convPE_(t)-E7-K3 vaccines,respectively. The TC-1 tumor suppression activity and cell mediatedimmune responses enhanced by these vaccines are examined using similarmethods as described above. Table 1 shows SEQ ID NOs. of the componentsof various fusion proteins. Table 2 shows the fusion proteins tested forthe effects on T cell-mediated immune responses in animals and thesequences of antigens.

TABLE 1 SEQ Length ID (resi- Component NO: dues) hCD28 Core  1  28TDIYFCKIEVMYPPPYLDNEKSNGTIIH hCD28 Maximum  2  53NCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDN EKSNGTIIHVKGPE_(t) Core (PE translocation domain core;   3  34a.a. 280- a.a. 313 of PE) PE_(t) Maximum (translocation domain maxi,   4112 a.a. 253- a.a. 364 of PE) RAP1 Minimum (domain III of RAP1)  5 104A2M Minimum  6 153 HIV-Tat Minimum  7  24HSPs Minimum, Heat shock 70 kDa protein   8 641 (HSPs; Homo sapiens)Minimum Pseudomonas exotoxin A (PE)    9 252binding domain Ia (an APC-binding  domain, a.a. 1- a.a. 252 of PE)Linker RXRXKR,), in which “X” is any   15   6 amino acid residue.Full length PE (Exotoxin A mature   10 613 form, Pseudomonas aeruginosa)Full length RAP1 (Homo sapiens low   11 323density lipoprotein receptor-related protein associated protein 1,LRPAP1); Domain 1: a.a. 1- a.a. 112;   domain 2: a.a. 113- a.a. 218;domain 3: a.a. 219- a.a. 323. Full length A2M (Homo sapiens alpha 2  12357 macroglobulin receptor-associated protein precursor)HIV-Tat (Human immunodeficiency virus 1) 13 101 KDEL 14   4 KKDLRDELKDEL16  12 KKDELRDELKDEL 17  13 KKDELRVELKDEL 18  13 KDELKDELKDEL 19  12PE₂₆₈₋₃₁₃ 20  46 PLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQV DQVIRCD28convPE_(t) 30  68 T¹D²I³Y⁴F⁵C⁶K⁷X⁸E⁹X¹⁰X¹¹Y¹²P¹³P¹⁴P¹⁵Y¹⁶X¹⁷D¹⁸N¹⁹E²⁰K²¹S²²N²³G²⁴T²⁵I²⁶I²⁷H²⁸R²⁹X³⁰R³¹X³²K³³R³⁴G³⁵W³⁶E³⁷Q³⁸L³⁹E⁴⁰Q⁴¹C⁴²G⁴³Y⁴⁴P⁴⁵V⁴⁶Q⁴⁷R⁴⁸L⁴⁹V⁵⁰A⁵¹L⁵²Y⁵³L⁵⁴A⁵⁵A⁵⁶R⁵⁷L⁵⁸S⁵⁹W⁶⁰N⁶¹Q⁶²V⁶³D⁶⁴Q⁶⁵V⁶⁶I⁶⁷R⁶⁸,wherein (X)⁸ is I or L; (X)¹⁰ is V, F or A, (X)¹¹ is M or L, X¹⁷ is L or I,(X)^(30,32) is any amino acid residue. CD28 consensus sequence 31  28T¹D²I³Y⁴F⁵C⁶K⁷(X)⁸E⁹(X)¹⁰(X)¹¹Y¹²P¹³P¹⁴P¹⁵Y¹⁶X¹⁷D¹⁸N¹⁸E²⁰K²¹S²²N²³G²⁴T²⁵I²⁶I²⁷ H²⁸,wherein (X)⁸ is I or L; (X)¹⁰ is V, F or A, (X)¹¹ is M or L, X¹⁷ is L or I. CD28 critical region 32  10K¹(X)²E³(X)⁴(X)⁵Y⁶P⁷P⁸P⁹Y¹⁰, wherein (X)² is I or L; (X)⁴ is V, F or A, (X)⁵ is M or L.

TABLE 2 Antigen SEQ ID Fusion protein name Antigen Name NO:RAP1-CD28convPE_(t)-E7-K3 HPV16 E7 (full length) 21RAP1-CD28convPE_(t)-E7₁₈-K3 HPV18 E7 (full length) 22RAP1-CD28convPE_(t)-HCVc-K3 HCV core protein (full length) 23RAP1-CD28convPE_(t)-HBx-K3 HBV X protein (full length) 24RAP1-CD28convPE_(t)-PCV2-K3 PCV2 ORF2 (a fragment 25 of ORF2)RAP1-CD28convPE_(t)-DGD-K3 PRRSV nucleocapsid 26 (a fusion antigen: ORF7a.a. 64-a.a. 123, linker and ORF7 a.a. 64-a.a. 123)RAP1-CD28convPE_(t)-M12-K3 PRRSV RNA-dependent 27 RNA polymerase (ORF1ba.a. 1046-a.a. 1210) RAP1-CD28convPE_(t)-PQAB-K3 PRRSV American strain:28 a fusion antigen of ORF6 (a.a. 2-a.a. 26) and ORF5 (a.a. 31-a.a. 63)RAP1-CD28convPE_(t)-RSAB-K3 PRRSV European strain: 29 a fusion antigenof ORF6 (a.a. 2-a.a. 28) and ORF5 (a.a. 31-a.a. 64)

In the immunogenicity assays, antigen-specific cell-mediated immuneresponses induced by various vaccines were evaluated by measuring thenumbers of CD3+/CD4+/IFNγ+ and CD3+/CD8+/IFNγ+ T cells in thesplenocytes. The results indicated that the vaccineRAP1-CD28convPE_(t)-antigen-K3 can induce strong T cell responses. FIG.14 B shows the CD3+/CD4+/IFNγ+ T cell number and the CD3+/CD8+/IFNγ+ Tcell number elicited by CD28convPE_(t)-E7_(˜)-K3 were about 50 times andgreater than 9 times of RAP1-K3, respectively.

The vaccine RAP1-CD28convPE_(t)-antigen-K3 is superior toPE₄₀₇-antigen-K3 in eliciting T cell-mediated immunogenicity. Forexample, FIG. 14A illustrates the CD3+/CD4+/IFNγ+ T cell number and theCD3+/CD8+/IFNγ+ T cell number elicited by CD28convPE₄-E7₁₆-K3 were about5 times and 7 times of PE₄₀₇-E7₁₆-K3, respectively. This indicates thatthe vaccine RAP1-CD28convPE_(t)-E7-K3 had a better cell-mediatedimmunogenicity than PE₄₀₇-E7-K3.

A fusion protein comprising RAP1 domain III, the sensitizing signalCD28conv alone without the translocation peptide PE_(t), antigen and anER retention signal is sufficient in eliciting a strong antigen-specificT cell mediated immune responses when the antigen chosen comprises tenor greater than 10 epitopes. FIG. 14C illustrates the vaccineRAP1-CD28conv-HCVcore-K3 elicited T cell responses with the numbers ofCD3+/CD4+/IFNγ+ and of CD3+/CD8+/IFNγ+ T cells being 20 times and 7.6times of the placebo group, respectively. The antigen HCVcore contains11 well-known MHC I epitopes.

It was unexpected that the ER retention signal is not essential for thefusion protein of the invention to elicit a strong cell-mediatedimmunogenicity. In other words, without the ER retention sequence, thefusion protein of the invention can still elicit strong T-cellresponses. FIG. 14D illustrates that the numbers of CD3+/CD4+/IFNγ+ andCD3+/CD8+/IFNγ+ T cells elicited by RAP1-CD28convPE_(t)-HBx (without theER retentions signal K3) were 7 times and 74 times of the placebo group.

In contrast, U.S. Pat. Nos. 7,378,100B2 and 7,335,361 show that the ERretention signal K3 is indispensable for PE-related fusion proteins(PE₄₀₇-antigen-K3) to elicit T cell responses.

It was also discovered that a fusion protein comprising RAP1 domain III,the translocation peptide PE₂₁₈₋₃₁₃ (without the sensitizing signalCD28conv), antigen and an ER retention signal is superior to aPE-related fusion protein without containing the RAP1 domain III. FIG.15C-J illustrate the vaccine RAP1-PE₂₆₈₋₃₁₃-PRRSV-K3 elicited greaterCD3+/CD4+/IFNγ+ and CD3+/CD8+/IFNγ+ T cell counts than the vaccinePE₄₀₇-PRRSV-K3.

The foregoing description of the exemplary embodiments of the inventionhas been presented only for the purposes of illustration and descriptionand is not intended to be exhaustive or to limit the invention to theprecise forms disclosed. Many modifications and variations are possiblein light of the above teaching. The embodiments and examples were chosenand described in order to explain the principles of the invention andtheir practical application so as to enable others skilled in the art toutilize the invention and various embodiments and with variousmodifications as are suited to the particular use contemplated.Alternative embodiments will become apparent to those skilled in the artto which the present invention pertains without departing from itsspirit and scope. Accordingly, the scope of the present invention isdefined by the appended claims rather than the foregoing description andthe exemplary embodiments described therein.

Some references, which may include patents, patent applications andvarious publications, are cited and discussed in the description of thisinvention. The citation and/or discussion of such references is providedmerely to clarify the description of the present invention and is not anadmission that any such reference is “prior art” to the inventiondescribed herein. All references cited and discussed in thisspecification are incorporated herein by reference in their entiretiesand to the same extent as if each reference was individuallyincorporated by reference.

1. A fusion protein comprising: (a) an antigen-presenting cell(APC)-binding domain or a CD91 receptor-binding domain, located at theN-terminus of the fusion protein; (b) a protein transduction domain,located at the C-terminus of the APC-binding domain or the CD91receptor-binding domain, wherein the protein transduction domain is afusion polypeptide consisting of: (1) a T cell sensitizingsignal-transducing peptide consisting of 28-53 amino acid residues inlength, comprising the amino acid sequence of SEQ ID NO: 31, in whichXaa⁸ is I; Xaa¹⁰ is V, F or A, Xaa¹¹ is M or L, Xaa¹⁷ is L or I, beinglocated at the N-terminus of the fusion polypeptide; (2) a translocationpeptide consisting of 34-112 amino acid residues in length, comprisingan amino acid sequence that is at least 90% identical to SEQ ID NO: 3,20 or 4; and (3) a linker, comprising SEQ ID NO: 15 linking the T cellsensitizing signal-transducing peptide and the translocation peptide;and (c) an antigen of a pathogen or a cancer cell, located at theC-terminus of the protein transduction domain; wherein: the pathogen isat least one selected from the group consisting of human papillomavirus(HPV), PRRSV, HIV-1, flu virus, Dengue virus, hepatitis C virus (HCV),hepatitis B virus (HBV), and porcine circovirus 2 (PCV2); and furtherwherein: the cancer cell is at least one selected from the groupconsisting of non-small cell lung cancer, breast carcinoma, melanoma,lymphomas, colon carcinoma, and hepatocellular carcinoma.
 2. The fusionprotein of claim 1, wherein the pathogen is at least one selected fromthe group consisting of HPV, PRRSV, HCV, HBV, and PCV2.
 3. The fusionprotein of claim 2, wherein the antigen of the pathogen is at least oneselected from the group consisting of HPV E7 protein, PRRSV protein, HCVcore protein, HBV X protein, and PCV2 ORF2 protein.
 4. The fusionprotein of claim 3, wherein the antigen comprises an amino acid sequencethat is at least 90% identical to SEQ ID NOs: 21, 22, 23, 24, 25, 26,27, 28 or
 29. 5. The fusion protein of claim 3, wherein the antigencomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 21, 22, 23, 24, 25, 26, 27, 28, and
 29. 6. The fusionprotein of claim 1, wherein the APC-binding domain or the CD91receptor-binding domain is a polypeptide comprising an amino acidsequence that is at least 90% identical to the sequence selected fromthe group consisting of SEQ ID NOs: 5, 9, 6, 7, and
 8. 7. The fusionprotein of claim 1, wherein the APC-binding domain or the CD91receptor-binding domain is a polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 5, 9, 6, 7,and
 8. 8. The fusion protein of claim 1, further comprising anendoplasmic reticulum retention sequence located at the C-terminus ofthe fusion protein.
 9. The fusion protein of claim 1, wherein the T cellsensitizing signal-transducing peptide comprises the amino acid sequenceof SEQ ID NO:
 1. 10. The fusion protein of claim 1, wherein thetranslocation peptide comprises the amino acid sequence of SEQ ID NO: 3.11. The fusion protein of claim 1, wherein the protein transductiondomain comprises the sequence of SEQ ID NO:
 30. 12. A fusion proteinconsisting of: (a) an antigen-presenting cell (APC)-binding domain or aCD91 receptor-binding domain, located at the N-terminus of the fusionprotein; (b) a protein transduction domain, located at the C-terminus ofthe APC-binding domain or the CD91 receptor-binding domain, wherein theprotein transduction domain is selected from the group consisting of:(i) a T cell-sensitizing signal-transducing peptide consisting of 28-53amino acid residues in length, comprising the amino acid sequence of SEQID NO: 31, in which Xaa⁸ is I; Xaa¹⁰ is V, F or A, Xaa¹¹ is M or L,Xaa¹⁷ is L or I; and (ii) a translocation peptide of 34-46 amino acidresidues in length, comprising an amino acid sequence that is at least90% identifical to SEQ ID NO: 3 or 20; and (c) an antigen of a pathogen,located at the C-terminus of the protein transduction domain; wherein:the APC-binding domain or the CD91 receptor-binding domain is free ofthe amino acid sequence of Pseudomonas exotoxin A (PE) binding domain Iif the protein transduction domain is the translocation peptide; andfurther wherein: the pathogen is at least one selected from the groupconsisting of human papillomavirus (HPV), PRRSV, HIV-1, flu virus,Dengue virus, hepatitis C virus (HCV), hepatitis B virus (HBV), andporcine circovirus 2 (PCV2), and the cancer cell is at least oneselected from the group consisting of non-small cell lung cancer, breastcarcinoma, melanoma, lymphomas, colon carcinoma, and hepatocellularcarcinoma.
 13. A fusion protein consisting of: (a) an antigen-presentingcell (APC)-binding domain or a CD91 receptor-binding domain, located atthe N-terminus of the fusion protein; (b) a protein transduction domain,located at the C-terminus of the APC-binding domain or the CD91receptor-binding domain, wherein the protein transduction domain isselected from the group consisting of: (i) a T cell-sensitizingsignal-transducing peptide consisting of 28-53 amino acid residues inlength, comprising the amino acid sequence of SEQ ID NO: 31, in whichXaa⁸ is I; Xaa¹⁰ is V, F or A, Xaa¹¹ is M or L, Xaa¹⁷ is L or I; and(ii) a translocation peptide of 34-46 amino acid residues in length,comprising an amino acid sequence that is at least 90% identical to SEQID NO: 3 or 20; and; (c) an antigen of a pathogen, located at theC-terminus of the protein transduction domain; and (d) an endoplasmicreticulum retention sequence located at the C-terminus of the fusionprotein; wherein: the pathogen is at least one selected from the groupconsisting of human papillomavirus (HPV), PRRSV, HIV-1, flu virus,Dengue virus, hepatitis C virus (HCV), hepatitis B virus (HBV), andporcine circovirus 2 (PCV2); and further wherein: the cancer cell is atleast one selected from the group consisting of non-small cell lungcancer, breast carcinoma, melanoma, lymphomas, colon carcinoma, andhepatocellular carcinoma.
 14. A fusion protein comprising: (a) anantigen-presenting cell (APC)-binding domain or a CD91 receptor-bindingdomain, located at the N-terminus of the fusion protein; (b) a proteintransduction domain, located at the C-terminus of the APC-binding domainor the CD91 receptor-binding domain, wherein the protein transductiondomain is a fusion polypeptide consisting of: (1) a T cell sensitizingsignal-transducing peptide of 28 amino acid residues in length,consisting of the amino acid sequence of SEQ ID NO: 31, in which Xaa⁸ isI or L; Xaa¹⁰ is V, F or A, Xaa¹¹ is M or L, Xaa¹⁷ is L or I, beinglocated at the N-terminus of the fusion polypeptide; (2) a translocationpeptide consisting of 34-112 amino acid residues in length, comprisingan amino acid sequence that is at least 90% identical to SEQ ID NO: 3,20 or 4; and (3) a linker, comprising SEQ ID NO: 15 linking the T cellsensitizing signal-transducing peptide and the translocation peptide;and (c) an antigen of a pathogen, located at the C-terminus of theprotein transduction domain; wherein: the pathogen is at least oneselected from the group consisting of human papillomavirus (HPV), PRRSV,HIV-1, flu virus, Dengue virus, hepatitis C virus (HCV), hepatitis Bvirus (HBV), and porcine circovirus 2 (PCV2); and further wherein: thecancer cell is at least one selected from the group consisting ofnon-small cell lung cancer, breast carcinoma, melanoma, lymphomas, coloncarcinoma, and hepatocellular carcinoma.
 15. The fusion protein of claim12, wherein the antigen comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 21, 22, 23, 24, 25, 26, 27, 28, and29.
 16. The fusion protein of claim 13, wherein the antigen comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:21, 22, 23, 24, 25, 26, 27, 28, and
 29. 17. The fusion protein of claim14, wherein the antigen comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 21, 22, 23, 24, 25, 26, 27, 28, and29.
 18. A method for inducing enhanced pathogen antigen-specific T cellresponses, comprising: administering a vaccine composition comprising atherapeutically effective amount of the fusion protein of claim 1 to asubject in need thereof, and thereby inducing enhanced pathogenantigen-specific T cell responses.
 19. A method for inducing enhancedpathogen antigen-specific T cell responses, comprising: administering avaccine composition comprising a therapeutically effective amount of thefusion protein of claim 13 to a subject in need thereof, and therebyinducing enhanced pathogen antigen-specific T cell responses.
 20. Amethod for inducing enhanced pathogen antigen-specific T cell responses,comprising: administering a vaccine composition comprising atherapeutically effective amount of the fusion protein of claim 14 to asubject in need thereof, and thereby inducing enhanced pathogenantigen-specific T cell responses.